RESUMO
Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.
Assuntos
Asma/patologia , Dermatophagoides pteronyssinus/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Semaforinas/genética , Actinas/genética , Actinas/metabolismo , Resistência das Vias Respiratórias/imunologia , Animais , Asma/imunologia , Modelos Animais de Doenças , Feminino , Células Caliciformes/imunologia , Imunoglobulina E/imunologia , Interleucina-10/genética , Contagem de Leucócitos , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , RNA Mensageiro/genética , Células Th17/imunologia , Células Th2/imunologiaRESUMO
Intracellular infection with the parasite Leishmania major features a state of concomitant immunity in which CD4+ T helper 1 (Th1) cell-mediated immunity against reinfection coincides with a chronic but sub-clinical primary infection. In this setting, the rapidity of the Th1 response at a secondary site of challenge in the skin represents the best correlate of parasite elimination and has been associated with a reversal in Leishmania-mediated modulation of monocytic host cells. Remarkably, the degree to which Th1 cells are absolutely reliant upon the time at which they interact with infected monocytes to mediate their protective effect has not been defined. In the present work, we report that CXCR3-dependent recruitment of Ly6C+ Th1 effector (Th1EFF) cells is indispensable for concomitant immunity and acute (<4 days post-infection) Th1EFF cell-phagocyte interactions are critical to prevent the establishment of a permissive pathogen niche, as evidenced by altered recruitment, gene expression and functional capacity of innate and adaptive immune cells at the site of secondary challenge. Surprisingly, provision of Th1EFF cells after establishment of the pathogen niche, even when Th1 cells were provided in large quantities, abrogated protection, Th1EFF cell accumulation and IFN-γ production, and iNOS production by inflammatory monocytes. These findings indicate that protective Th1 immunity is critically dependent on activation of permissive phagocytic host cells by preactivated Th1EFF cells at the time of infection.
Assuntos
Imunidade Celular/imunologia , Leishmaniose Cutânea/imunologia , Monócitos/imunologia , Células Th1/imunologia , Animais , Leishmania major/imunologia , Camundongos Endogâmicos C57BLRESUMO
There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.
Assuntos
Leishmania major/metabolismo , Leishmania major/patogenicidade , Leishmaniose Cutânea/parasitologia , Fosfoenolpiruvato/metabolismo , Fatores de Virulência/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Citocinas/imunologia , Feminino , Imunidade Celular/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Fosfoenolpiruvato/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Fatores de Virulência/imunologiaRESUMO
Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05-0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.
Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/metabolismo , Semaforinas/metabolismo , Células Th1/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Humanos , Tolerância Imunológica , Leishmaniose Cutânea/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Semaforinas/genéticaRESUMO
Inflammation is critical for controlling pathogens, but also responsible for symptoms of infectious diseases. IL-27 is an important regulator of inflammation and can limit development of IFNγ-producing Tbet+ CD4+ T (Th1) cells. IL-27 is thought to do this by stimulating IL-10 production by CD4+ T cells, but the underlying mechanisms of these immunoregulatory pathways are not clear. Here we studied the role of IL-27 signalling in experimental visceral leishmaniasis (VL) caused by infection of C57BL/6 mice with the human pathogen Leishmania donovani. We found IL-27 signalling was critical for the development of IL-10-producing Th1 (Tr1) cells during infection. Furthermore, in the absence of IL-27 signalling, there was improved control of parasite growth, but accelerated splenic pathology characterised by the loss of marginal zone macrophages. Critically, we discovered that IL-27 signalling limited glycolysis in Th1 cells during infection that in turn attenuated inflammation. Furthermore, the modulation of glycolysis in the absence of IL-27 signalling restricted tissue pathology without compromising anti-parasitic immunity. Together, these findings identify a novel mechanism by which IL-27 mediates immune regulation during disease by regulating cellular metabolism.
Assuntos
Interleucinas/metabolismo , Leishmaniose Visceral/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Glicólise , Interferon gama/imunologia , Interleucinas/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Baço/imunologiaRESUMO
Semaphorin 3E (Sema3E) is a secreted protein that was initially discovered as a neuronal guidance cue. Recent evidence showed that Sema3E plays an essential role in regulating the activities of various immune cells. However, the exact role of Sema3E in macrophage function, particularly during inflammation, is not fully understood. We studied the impact of Sema3E gene deletion on macrophage function during the LPS-induced acute inflammatory response. We found that Sema3E-deficient (Sema3e-/- ) mice were better protected from LPS-induced acute inflammation as exemplified by their superior clinical score and effective temperature control compared with their wild-type littermates. This superior control of inflammatory response in Sema3e-/- mice was associated with significantly lower phosphorylation of ERK1/2, AKT, STAT3, and NF-κB, and a concomitant reduction in inducible NO synthase expression and production of TNF and IL-6 compared with their Sema3e+/+ littermates. Sema3e-/- mice also contained significantly higher numbers of activated macrophages compared with their Sema3e+/+ littermates at both baselines and after LPS challenge. In vivo-specific deletion of the Sema3E high-affinity receptor, plexinD1, on macrophages led to the improvement in clinical disease following exposure to a lethal dose of LPS. Collectively, our data show that Sema3E plays an essential role in dampening the early inflammatory response to LPS by regulating macrophage function, suggesting an essential role of this pathway in macrophage inflammatory response.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Semaforinas/imunologia , Animais , Células Cultivadas , Inflamação/induzido quimicamente , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Semaforinas/deficiênciaRESUMO
There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Di-Hidrolipoamida Desidrogenase/imunologia , Leishmania/imunologia , Animais , Linhagem Celular , Feminino , Interferon gama/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases, their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-γ and TNF-α; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article, we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia, which was accompanied by significant reduction in IFN-γ production by immune cells in the spleens and liver of infected mice. Strikingly, infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-γ and TNF-α) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively, these studies show that NK cells are critical for optimal resistance to T. congolense, and its deficiency leads to enhanced susceptibility in infected mice.
Assuntos
Células Matadoras Naturais/imunologia , Tripanossomíase Africana/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Trypanosoma congolense/imunologiaRESUMO
The outcome of intracellular parasitic infection can be determined by the immunoregulatory activities of natural regulatory CD4+ Foxp3+ T (Treg) cells and the anti-inflammatory cytokine IL-10. These mechanisms protect tissue but can also suppress antiparasitic CD4+ T cell responses. The specific contribution of these regulatory pathways during human parasitic diseases remains unclear. In this study, we investigated the roles of Treg cells and IL-10 during experimental visceral leishmaniasis caused by Leishmania donovani infection of C57BL/6 mice. We report only a limited contribution of Treg cells in suppressing antiparasitic immunity, but important roles in delaying the development of splenic pathology and restricting leukocyte expansion. We next employed a range of cell-specific, IL-10- and IL-10R-deficient mice and found these Treg cell functions were independent of IL-10. Instead, conventional CD4+ T cells and dendritic cells were the most important cellular sources of IL-10, and the absence of IL-10 in either cell population resulted in greater control of parasite growth but also caused accelerated breakdown in splenic microarchitecture. We also found that T cells, dendritic cells, and other myeloid cells were the main IL-10-responding cells because in the absence of IL-10R expression by these cell populations, there was greater expansion of parasite-specific CD4+ T cell responses associated with improved control of parasite growth. Again, however, there was also an accelerated breakdown in splenic microarchitecture in these animals. Together, these findings identify distinct, cell-specific, immunoregulatory networks established during experimental visceral leishmaniasis that could be manipulated for clinical advantage.
Assuntos
Interleucina-10/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD4/metabolismo , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunomodulação , Camundongos , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.
Assuntos
Alternativas aos Testes com Animais/métodos , Antiprotozoários/toxicidade , Citometria de Fluxo/métodos , Leishmania/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , Adulto , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Antimoniato de Meglumina/toxicidade , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Fatores de Tempo , Adulto JovemRESUMO
In their quiescent state, Hepatic stellate cells (HSCs), are present in the sub-endothelial space of Disse and have minimal interaction with immune cells. However, upon activation following injury, HSCs directly or indirectly interact with various immune cells that enter the space of Disse and thereby regulate diverse hepatic function and immune physiology. Other than the normal physiological functions of HSCs such as hepatic homeostasis, maturation and differentiation, they also participate in hepatic inflammation by releasing a battery of inflammatory cytokines and chemokines and interacting with other liver cells. Here, we have reviewed the role of HSC in the pathogenesis of liver inflammation and some infectious diseases in order to understand how the interplay between immune cells and HSCs regulates the overall outcome and disease pathology.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Células Estreladas do Fígado/imunologia , Fígado/imunologia , Animais , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Hepatite/imunologia , Hepatite/metabolismo , Hepatite/virologia , Humanos , Fígado/microbiologia , Fígado/patologia , Fígado/virologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Despite decades of clinical and biomedical research, the pathogenesis of sepsis and its spectrum of diseases (severe sepsis and septic shock), which are leading causes of death in intensive care units, are still poorly understood. In this article, we show that signaling via the p110δ isoform of PI3K is critical for survival in experimental sepsis. Mice with an inactive knock-in mutation in the p110δ gene (p110δD910A) succumbed acutely to nonlethal dose LPS challenge. The susceptibility of p110δD910A mice to LPS was associated with increased neutrophil numbers and activities in the tissues, due in part to delayed apoptosis resulting mostly from inherent reduced regulatory T cell (Treg) numbers. Adoptive transfer of wild-type or p110δD910A Tregs abrogated exaggerated neutrophil activity, increased neutrophil apoptosis, and rescued p110δD910A mice from mortality after LPS challenge. We confirmed the clinical relevance of these findings by showing that human Tregs also regulate neutrophil function and survival. Collectively, our results show that PI3K δ is essential for survival during sepsis. In addition, our data highlight the importance of Tregs in regulating the pathogenesis of sepsis and septic shock via their effects on neutrophil survival and function, and provide evidence of regulation of innate immunity by cells of the adaptive immune system.
Assuntos
Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Sepse/imunologia , Choque Séptico/mortalidade , Linfócitos T Reguladores/imunologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Classe I de Fosfatidilinositol 3-Quinases , Técnicas de Introdução de Genes , Imunidade Inata , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Neutrófilos/patologia , Neutrófilos/fisiologia , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Isoformas de Proteínas , Sepse/fisiopatologia , Choque Séptico/imunologia , Choque Séptico/fisiopatologia , Transdução de SinaisRESUMO
Semaphorins are an essential family of guidance cues ubiquitously expressed in various organs, which play diverse developmental, homeostatic, and pathological roles. Semaphorin 3E (Sema3E), initially identified as a neuronal chemorepellent, is involved in the regulation of cell migration, proliferation, and angiogenesis. However, expression and function of Sema3E in allergic asthma has not been extensively investigated. We determined the expression of Sema3E in the airways and its effect on airway inflammation, hyperresponsiveness, and remodeling as pathological features of allergic asthma provoked by house dust mite in vivo. Our data indicate that exposure to house dust mite markedly reduces Sema3E expression in mouse airways. More important, replenishment of Sema3E by intranasal administration of exogenous Sema3E protects mice from allergic asthma by reducing eosinophilic inflammation, serum IgE level, and T helper cell 2/T helper cell 17 cytokine response. The regulatory effect of Sema3E on cytokine response was sustained on allergen recall response in the lymph nodes and spleen. Furthermore, goblet cell hyperplasia, collagen deposition, and airway hyperresponsiveness were significantly diminished on Sema3E treatment. The inhibitory effect of Sema3E was associated with a reduction of pulmonary CD11b+ conventional dendritic cells and regulation of CD4+ T-cell cytokine response. Collectively, our data represent a novel approach to treating allergic asthma via regulation of immune response to house dust mite.
Assuntos
Asma/prevenção & controle , Regulação da Expressão Gênica , Glicoproteínas/administração & dosagem , Proteínas de Membrana/administração & dosagem , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/prevenção & controle , Administração Intranasal , Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/imunologia , Alérgenos/imunologia , Animais , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Proteínas do Citoesqueleto , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Inflamação/imunologia , Inflamação/prevenção & controle , Pulmão/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Hipersensibilidade Respiratória/imunologia , Semaforinas , Células Th17/imunologia , Células Th2/imunologiaRESUMO
OBJECTIVES: This study aimed to evaluate the immuno-prophylactic and -therapeutic effect of p110δ-specific pharmacological inhibitors (CAL-101 and IC87114), either alone or in combination with amphotericin B, against experimental cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). METHODS: Female BALB/c mice were infected intravenously with Leishmania donovani or subcutaneously with Leishmania major Prophylactic treatment was initiated 24 hâ prior to infection, whereas therapeutic treatments with or without amphotericin B were initiated either 1â week or 2â weeks post-infection. At different times post-infection, mice were sacrificed and parasite burden, regulatory T cell (Treg) numbers and cytokine production were assessed in the liver, spleen, draining lymph nodes and footpads. In addition, direct cytolytic effects of the inhibitors on parasite growth in axenic cultures and inside infected and uninfected macrophages were also assessed. RESULTS: Prophylactic and therapeutic administration of p110δ pharmacological inhibitors significantly reduced cutaneous lesion (in CL) and parasite burdens (in VL and CL) in the spleens, livers and footpads of infected mice. The reduction in parasite burden was associated with a concomitant reduction in Treg numbers and cytokine production by liver, spleen and lymph node cells. Combined low-dose CAL-101 and amphotericin B therapy caused complete clearance of parasites in mice infected with L. donovani CONCLUSIONS: Our studies clearly show a novel therapeutic option for leishmaniasis based on CAL-101 monotherapy or CAL-101 and amphotericin B combination therapy. These observations have important and direct implications for antimicrobial immunotherapy and drug/vaccine development against leishmaniasis.
Assuntos
Leishmania donovani/efeitos dos fármacos , Leishmania donovani/imunologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Linfócitos T Reguladores/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Anfotericina B/uso terapêutico , Animais , Contagem de Linfócito CD4 , Classe I de Fosfatidilinositol 3-Quinases , Citocinas/biossíntese , Quimioterapia Combinada , Feminino , Imunomodulação/efeitos dos fármacos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Fígado/metabolismo , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Purinas/farmacologia , Quinazolinas/farmacologia , Quinazolinonas/farmacologia , Baço/metabolismoRESUMO
UNLABELLED: Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δ(D910A) ) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-ß, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani-infected HSCs induce CD4(+) T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. CONCLUSION: These results demonstrate the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/imunologia , Células Estreladas do Fígado/imunologia , Imunidade Celular/imunologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Although some studies indicate that the interaction of CD40 and CD40L is critical for IL-12 production and resistance to cutaneous leishmaniasis, others suggest that this pathway may be dispensable. In this article, we compared the outcome of Leishmania major infection in both CD40- and CD40L-deficient mice after treatment with rIL-12. We show that although CD40 and CD40L knockout (KO) mice are highly susceptible to L. major, treatment with rIL-12 during the first 2 wk of infection causes resolution of cutaneous lesions and control of parasite replication. Interestingly, although treated CD40 KO mice remained healed, developed long-term immunity, and were resistant to secondary L. major challenge, treated CD40L KO reactivated their lesion after cessation of rIL-12 treatment. Disease reactivation in CD40L KO mice was associated with impaired IL-12 and IFN-γ production and a concomitant increase in IL-4 production by cells from lymph nodes draining the infection site. We show that IL-12 production by dendritic cells and macrophages via CD40L-macrophage Ag 1 (Mac-1) interaction is responsible for the sustained resistance in CD40 KO mice after cessation of rIL-12 treatment. Blockade of CD40L-Mac-1 interaction with anti-Mac-1 mAb led to spontaneous disease reactivation in healed CD40 KO mice, which was associated with impaired IFN-γ response and loss of infection-induced immunity after secondary L. major challenge. Collectively, our data reveal a novel role of CD40L-Mac-1 interaction in IL-12 production, development, and maintenance of optimal Th1 immunity in mice infected with L. major.
Assuntos
Antígenos CD40/genética , Ligante de CD40/genética , Subunidade p35 da Interleucina-12/farmacologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Feminino , Interferon gama/biossíntese , Interferon gama/imunologia , Subunidade p35 da Interleucina-12/biossíntese , Subunidade p35 da Interleucina-12/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Linfonodos/citologia , Linfonodos/imunologia , Antígeno de Macrófago 1/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/farmacologia , Recidiva , Células Th1/imunologiaRESUMO
We previously showed that LIGHT and its receptor herpes virus entry mediator (HVEM) are important for development of optimal CD4(+) Th1 cell immunity and resistance to primary Leishmania major infection in mice. In this study, we further characterized the contributions of this molecule in dendritic cell (DC) maturation, initiation, and maintenance of primary immunity and secondary anti-Leishmania immunity. Flow-cytometric studies showed that CD8α(+) DC subset was mostly affected by HVEM-Ig and lymphotoxin ß receptor-Ig treatment. LIGHT signaling is required at both the priming and the maintenance stages of primary anti-Leishmania immunity but is completely dispensable during secondary immunity in wild type mice. However, LIGHT blockade led to impaired IL-12 and IFN-γ responses and loss of resistance in healed CD40-deficient mice after L. major challenge. The protective effect of LIGHT was mediated primarily via its interaction with lymphotoxin ß receptor on CD8α(+) DCs. Collectively, our results show that although LIGHT is critical for maintenance of primary Th1 response, it is dispensable during secondary anti-Leishmania immunity in the presence of functional CD40 signaling as seen in wild type mice.
Assuntos
Antígenos CD40/deficiência , Interações Hospedeiro-Patógeno/imunologia , Leishmaniose Cutânea/imunologia , Células Th1/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Feminino , Regulação da Expressão Gênica , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/imunologia , Camundongos , Transdução de Sinais , Células Th1/parasitologia , Células Th1/patologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity.
Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Proteínas/imunologia , Células Th1/citologia , Células Th1/imunologia , Animais , Diferenciação Celular/imunologia , Proliferação de Células , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/imunologia , Suscetibilidade a Doenças/imunologia , Imunidade Celular/imunologia , Interferon gama/biossíntese , Leishmaniose Cutânea/parasitologia , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Óxido Nítrico/biossíntese , Ovalbumina , Proteínas/genética , Células Th17/citologia , Células Th17/imunologia , Células Th2/citologia , Células Th2/imunologiaRESUMO
Leishmaniasis affects millions of people worldwide and continues to pose public health problem. There is extensive evidence supporting the critical role for IL-12 in initiating and maintaining protective immune response to Leishmania infection. Although gene deletion studies show that CD40-CD40L interaction is an important pathway for IL-12 production by antigen-presenting cells and subsequent development of protective immunity in cutaneous leishmaniasis, several studies have uncovered other pathways that could also lead to IL-12 production and immunity in the absence of intact CD40-CD40L signaling. Here, we review the literature on the role of IL-12 in the induction and maintenance of protective T cell-mediated immunity in cutaneous leishmaniasis and the different pathways leading to IL-12 production by antigen-presenting cells following Leishmania major infection.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Imunidade Celular , Interleucina-12/metabolismo , Leishmaniose Cutânea/imunologia , Transdução de Sinais , Células Th1/imunologia , Animais , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/metabolismo , Técnicas de Inativação de Genes , Humanos , Terapia de Alvo MolecularRESUMO
Regulatory T cells (Tregs) are essential for maintenance of peripheral tolerance, and defects in Treg function have been linked to several autoimmune diseases. We previously reported that depletion of Tregs resulted in mortality to an otherwise nonlethal dose of LPS or Escherichia coli challenge. In this study, we investigated the mechanism by which Treg depletion leads to enhanced susceptibility to LPS. Using different murine lymphocyte gene knockout models, we show that the enhanced sensitivity to LPS following Treg depletion is mediated by T cells. SCID or RAG1-deficient mice, which lack T and B cells, do not show enhanced susceptibility to LPS. However, reconstitution of SCID mice with wild-type CD4(+) T cells restored Treg depletion-induced sensitivity to LPS. This CD4(+) T cell-mediated hypersensitivity to LPS challenge in the absence of Tregs was also observed upon reconstitution of SCID mice with CD4(+) T cells from CD25 knockout mice (which lack functional Tregs). Additionally, depletion of Tregs leads to increased CD4(+) T cell proliferation and proinflammatory cytokine production in response to LPS challenge. Some CD4(+) T cells express TLR4, and pretreatment of CD4(+) T cells with LPS dramatically enhanced their ability to induce inflammatory cytokine production by macrophages. Collectively, our results indicate that in the absence of functional Tregs, CD4(+) T cells are pathologic and contribute to exaggerated immune activation that is detrimental for survival in LPS-induced acute inflammation. Our data also provide evidence for direct activation of CD4(+) T cells by LPS through TLR4.